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First published online June 15, 2007
Journal of Experimental Biology 210, 2278-2289 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.004770

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Identification and cardiotropic actions of sulfakinin peptides in the American lobster Homarus americanus

Patsy S. Dickinson^1,*, Jake S. Stevens¹, Szymon Rus¹, Henry R. Brennan¹, Christopher C. Goiney², Christine M. Smith³, Lingjun Li^4,5, David W. Towle³ and Andrew E. Christie^2,3

¹ Department of Biology, Bowdoin College, 6500 College Station, Brunswick, ME 04011, USA
² Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA
³ Mount Desert Island Biological Laboratory, PO Box 35, Old Bar Harbor Road, Salisbury Cove, ME 04672, USA
⁴ School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705-2222, USA
⁵ Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706-1396, USA

View larger version (50K): [in this window] [in a new window]   Fig. 1. Nucleotide and deduced amino acid sequences of Homarus americanus prepro-sulfakinin. Within the coding region of the cDNA (bold font), both the start (ATG) and stop (TAA) codons are underlined, as are the two polyadenylation signal sequences (AATAA) present in the 3'-UTR. The predicted amino acid sequence of the prepro-hormone signal peptide is shown in italics. Predicted cleavage sites within the preprohormone are shown in black. The amino acid sequence, including the C-terminal glycine residue which serves as a target for -amidation, of Homarus americanus sulfakinin I (Hoa-SK I) is shown in blue, while that of Homarus americanus sulfakinin II (Hoa-SK II), including its C-terminal glycine, is shown in red. The amino acid sequences of three putatative Homarus americanus sulfakinin-precursor related peptides [Hoa-SPRPs I, II and III (named based on their relative positions within the prepro-hormone)] are shown in green (including the C-terminal glycine residue of Hoa-SPRP II). Within the amino acid sequence of the prepro-hormone, the position of the stop codon is denoted with an asterisk.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Flow diagram showing the putative post-translational processing of sulfakinins and sulfakinin precursor-related peptides (SPRPs) from the deduced Homarus americanus prepro-sulfakinin. Translation of the nucleotide sequence of the cDNA encoding H. americanus prepro-sulfakinin predicts a 120 amino acid prepro-hormone (top sequence). The first 24 amino acids of the prepro-hormone are predicted to be a signal peptide (SignalP 3.0 analysis) (Bendtsen et al., 2004), with a cleavage site between Ser24 and Ala25 (red residues and arrowhead). Processing between these residues by signal peptidase would produce a 96 amino acid pro-sulfakinin (second sequence). Via homology to known insect pro-hormone cleavage sites (Veenstra, 2000), two Lys-Arg and two Arg-Xn-Arg (where X is a variable amino acid and n is either 0, 2, 4 or 6 residues) processing sites were identified in Hoa-pro-sulfakinin (yellow residues and arrowheads). Proteolytic processing by a prohormone convertase at these sites would liberate five peptides (third line of sequences); the basic residues on four of these are predicted to be the targets of carboxypeptidase action (green residues and arrowheads). In three of these four peptides, carboxypeptidase action would expose a glycine residue (fourth line of sequences), which likely serves as a target for -amidation by peptidyl-amidating monooxygenase [blue residues and arrowheads; homology to known sulfakinin isoforms (e.g. Johnsen et al., 2000; Torfs et al., 2002)]. Action by this enzyme would result in the amidation of the carboxy termini of these three peptides (fifth line of sequences). Additional post-translational processing of tyrosine residues by tyrosylprotein sulfotransferase in two of the peptides is predicted to result in the addition of sulfate groups to them [purple residues and arrowheads; homology to known sulfakinin isoforms (e.g. Johnsen et al., 2000; Torfs et al., 2002) and prediction via Sulfinator software (Monigatti et al., 2002)]. Likewise, based on homology to known sulfakinin isoforms (e.g. Johnsen et al., 2000; Torfs et al., 2002), the amino (N)-terminal glutamic acid in one peptide and the N-terminal glutamine in another (purple residues and arrowheads) are hypothesized to undergo enzymatic or spontaneous cyclization, resulting in the formation of pyro-residues in the mature forms (final line of sequences). The five resulting peptides (two SKs and three SPRPs) are shown and labeled in white.

View larger version (106K): [in this window] [in a new window]   Fig. 3. Both Hoa-SK I and Hoa-SK II (10–6 mol l-1) enhanced activity of the isolated heart. Shown are recordings from a force-displacement transducer in two preparations. (A,B) Recordings from a preparation in which the heartbeat was very regular. Each of the peptides evoked an increase in both frequency and amplitude of the heartbeat, although the effects were more pronounced with Hoa-SK II than with Hoa-SK I. (C,D) Recordings from a second preparation in which the heartbeat showed periodically irregularities. In addition to increasing the frequency and amplitude of heart contractions, Hoa-SK I and Hoa-SK II regularized heart beat frequency and amplitude when the heartbeat was initially irregular. Bar, 10 s for all recordings.

View larger version (7K): [in this window] [in a new window]   Fig. 4. Hoa-SK I and Hoa-SK II evoked increases in the frequency and amplitude of heart contractions. Both frequency and amplitude of cardiac contractions in the isolated heart increased by approximately 15% when the hearts were perfused with Hoa-SK I; frequency increased on average by 20% and amplitude by 25% when the hearts were perfused with Hoa-SK II. All changes were significantly different from 0 (two-tailed t-tests): *P<0.05; **P<0.01; N=11 preparations for Hoa-SK I, N=14 preparations for Hoa-SK II. Values are means ± s.e.m.

View larger version (23K): [in this window] [in a new window]   Fig. 5. The increases in both frequency and amplitude of heartbeat induced by Hoa-SK I (A,C) and Hoa-SK II (B,D) were rapid in onset, and relatively rapid in time to return to baseline. Shown are examples from a single, representative preparation, in which it can be seen that the peak effect was reached within less than 5 min. Wash-out took somewhat longer, but activity had returned to baseline within 10–12 min.